脂多糖刺激单核巨噬细胞培养上清液对成骨细胞成骨特性的影响

Effects of LPS-stimulated monocyte culture supernatant on the osteogenic function of osteoblastic cells in vitro

目的探讨建立牙周炎症的体外实验模型的可行性，为后续进行牙周炎症状态下应力对牙槽骨改建的研究奠定基础。方法以牙龈卟啉单胞菌（Porphyromonas gingivalis，P．g）脂多糖（1ipopolysaccharide，LPS）Pg～I．PS刺激小鼠单核巨噬细胞（RAW264．7）的培养上清液作用于体外培养的小鼠成骨细胞（MC3T3E1）24h和48h后，分别通过噻唑蓝（MTT）法和碱性磷酸酶（ALP）试剂盒检测MC3T3E1的增殖活性和碱性磷酸酶活性，观察脂多糖刺激的小鼠单核巨噬细胞培养上清液对小鼠成骨细胞成骨功能的影响。结果Pg-I，PS刺激RAW264．7后其上清液含I，18、IL-6和TNF-α等炎症因子，且与LPS的浓度和刺激时间呈依赖性。此上清液对MC3T3-E1的增殖和ALP活性均有抑制作用，亦与上清液中以上炎症因子的浓度呈依赖性，与体内牙周炎症状态相似。结论PgLPS体外刺激RAW264．7的培养上清液可以应用于建立体外牙周炎症模型，这将为后续研究牙周炎症状态下应力对牙槽骨改建的影响奠定初步的基础。

Objective To investigate the influence of Pg-I.PS stimulated monocyte （RAW 264.7） culture supernatant on the proliferation and alkaline phosphatase （ALP） activity of osteoblastic cells （MC3T3 El） in vitro. Methods The culture supernatant of monocytes stimulated with Pg-LPS was applied to osteoblasts MC3T3 E1 with different diluted concentrations（ 10%, 20 %, 30 %, 40 % and 50 %） simultaneously for 24 h and 48 h in vitro. The cellular proliferation was assessed by MTT assay, Cellular AI.P activity was examined using ALP measurement kit, and the results were statistically analyzed using SPSS 12.0 software package. Results LPS stimulated RAW264.7 to release inflammatory cytokines including IL-1β, IL-6 and TNF-α, whose secretion was in a time and dose dependent manner with the concentration of LPS. After the stimulation of different concentrations of inflammatory supernatant, the proliferation and ALP activity of MC3T3-E1 cells significantly deceased, both in a concentration-dependent manner. Conclusions These results indicated that the inflammatory cytokine secretion of Pg-LPS stimulated RAW264. 7 culture supernatant was similar to the change in vivo with inflammation. The inflammatory supernatant could inhibit the proliferation and ALP activity of osteoblastic cells.